The latest decades have experienced a rapid development of new methods for measurement of reverse transcription, RNA dependent DNA polymerization. The more complex issue concerning methods for quantification of DNA dependent DNA polymerization has so far received much less attention.
The classic DNA polymerase activity assays involve use of DNAse treated DNA (“activated DNA”) as primer/template and incorporation of radiolabeled nucleotides into DNA (Aposhian and Komberg 1962). Measurement of acid preciptable radioactivity allows calculation of the amount of nucleotides incorporated and the number of enzyme units present. However, use of radioactivity is currently restricted and discouraged in many laboratories and there is due to this a general trend away from radioactivity-based techniques.
For DNA polymerases a commercial assay based on ELISA detection of digoxigenine labeled nucleotides incorporated in newly made DNA is available (Roche Molecular Biochemicals Cat. no 1468120, U.S. Pat. No. 5,635,350). This assay is hampered by the use of two different nucleotide substrate analogues with bulky groups, digoxigenine as label and biotin for product immobilization. As a result the polymerization reaction velocity and subsequent detection sensitivity is reduced. The utilization of substrate analogues with highly deviating kinetic properties makes this system less relevant for studies of drug susceptibility of different polymerases.
Another more attractive alternative method is a fluorescence-based assay for DNA polymerase holoenzyme, based on the specific reaction of the dye PicoGreen with double-stranded DNA (Seville et all 1996). The latter process has recently been modified to make it suitable for a broader range of different DNA polymerizing enzymes (Tveit and Kristensen 2001). This assay is technically simple and based on the utilization of natural nucleotides. The detection sensitivity is, however, still in the same range as the classic radioactive DNA polymerase assay and the applications described demonstrates a detection range of 0.05-0.5 U DNA polymerase/sample.
HIV therapy today is based on multidrug therapy. The regimens are based on combinations of all three types of drugs available: nucleoside analogues, non-nucleoside analogues and protease inhibitors. The strategy is to minimize the probability for a mutant virus to survive.
The reverse transcriptase (RT) inhibitors are either nucleoside analogues or non-nucleoside analogues. The non-nucleoside inhibitors bind to a hydrophobic pocket in the RT enzyme close to, but not contiguous with, the active site. HIV-1 replication is inhibited allosterically by displacing the catalytic aspartate residues relative to the polymerase binding site.
The nucleoside inhibitors used today terminates the DNA chain elongation as they lack a 3′-hydroxyl group. Prolonged therapy with nucleoside inhibitors commonly leads to the development of resistant virus. This process is associated with the gradual appearance of mutations in the virus pol gene, each leading to defined amino acid substitutions (for a review see Vandamme et al 1998). The effects of these substitutions at the enzymatic levels is complicated and includes enhancement of a primitive DNA editing function. This reaction is nucleotide dependent and produces dinucleoside polyphosphate and an extendible DNA 3′ end (Arion et al 1998, Meyer et al 1999).
The HIV-1 RT as well as other reverse transcriptases perform three different enzymatic reactions: RNA-dependent DNA polymerization, DNA-dependent DNA polymerization, and degradation of RNA in the DNA-RNA hybrid (RNase H). The HIV reverse transcriptase, encoded by the pol gene, is a heterodimer consisting of a p66 and a p51 subunit. Both RNA-dependent DNA polymerization and DNA-dependent DNA polymerization are performed by the same active site localized in the p66 subunit (for a review see Goff 1990). The reaction mechanism of these drugs has mainly been defined according to their action on the RNA-dependent DNA polymerization reaction. The effect on the DNA-dependent DNA polymerization reaction is comparatively less studied.
Provided that the reaction mechanism and the active metabolized drug is known and available, phenotypic virus drug susceptibility could be determined at the enzyme level. Depending on the capacity of the enzyme assays and the virus isolation techniques used, the drug sensitivity testing can theoretically be done either on supernatants from virus culture propagation, the primary virus isolation or on virus preparations recovered directly from the patients. Conventional RT activity assay is performed by utilizing an artificial template-primer construction and labeled deoxynucleoside triphosphate as nucleotide substrate. The template/primer pair poly(rA)/oligo(dT) is the most efficient and most used combination for determination of HIV as well as for other retroviral RTs. A drawback of this type of assay when drug sensitivity testing is concerned, is that only non-nucleoside analogues or analogues that can base pair with rA can be tested. Analogues to the other nucleotide bases will require an assay based on a variable polymer template. RNA polymers containing pyrimidine bases are notoriously sensitive to RNases and in practice not compatible with biological samples. It would therefore be advantageous to base a polymerase assay intended for drug sensitivity testing on a variable DNA template, provided that the assay system gives results that correlate with those from inhibition of reverse transcription and classic phenotypic drug resistance tests.
HIV therapy used today is only one example of the potency of DNA polymerase inhibitors. The current situation concerning resistance development among bacteria and other microorganisms motivates the search for new classes of antimicrobial drugs. DNA polymerases are one of the major targets during this effort. As such there is a great demand for technically simple polymerase assays, which do not cause potential environmental hazards and can be applied for drug screening towards a broad range of microbial DNA polymerase isozymes. The toxicity of the drug leads found must further be evaluated towards the corresponding mammalian DNA polymerases.
Quantification of proliferation associated polymerases such as polymerase-α and -δ can be used for monitoring cell proliferation. It may be mentioned in this context that the serum levels of thymidine kinase, another cell proliferation associated enzyme, are currently used for prognosis and classification of malignant disease (U.S. Pat. No. 4,637,977). Phosphorylation of thymidine is just one of the two intracellular synthetic pathways, which provides thymidine triphosphate for DNA synthesis. Measurement of the DNA polymerase itself has the potential to give a more correct estimation of total DNA synthesis compared to thymidine kinase activity or thymidine incorporation.